JP6115995B2 - Non-enzymatic saccharification reaction inhibitor - Google Patents

Description

  The present invention relates to a non-enzymatic saccharification reaction inhibitor that can effectively inhibit a non-enzymatic saccharification reaction of a protein in which a protein and a sugar are non-enzymatically bound.

  When an amino group present in a protein and an aldehyde group of a reducing sugar such as glucose react non-enzymatically, a glycated product is formed. This reaction is called non-enzymatic saccharification reaction or Maillard reaction, and has been used in the field of food chemistry for a long time. Non-enzymatic saccharification reactions occur in vivo, and glycated proteins are always formed. The non-enzymatic saccharification reaction is considered to occur in two stages, a first-stage reaction in which an Amadori rearrangement product is formed via a Schiff base and a second-stage reaction in which complex cleavage or condensation occurs. In the second-stage reaction, AGEs (= A variety of non-single substances are produced, called Advanced Glycation End Products.

  It has been pointed out that AGEs may act as a factor that develops and develops symptoms, diseases (diseases), etc. that occur in relation to non-enzymatic glycation reactions of proteins. For example, in Non-Patent Documents 1 to 3, AGEs accumulate in the living body with aging, and are related to various tissue disorders recognized by aging, for example, hardening of connective tissues such as skin, blood vessel walls, joints, etc. It has been disclosed that it can cause thickening of the skin, formation of wrinkles, arteriosclerosis, arthritis and the like. Non-Patent Document 4 discloses that AGEs are related to cataracts. In addition, it has been pointed out that it not only causes tissue damage due to aging, but is also involved in diseases such as sunlight elastic fibrosis, Alzheimer's disease, diabetic complications, etc. Discloses that Nε- (carboxymethyl) lysine (CML), a kind of AGEs, has been detected in the skin tissue of sunlight elastic fibrosis, and the accumulation of CML is disclosed. It is suggested to be involved in lesions such as deep wrinkles and skin tissue hardening. Non-Patent Document 6 discloses that AGEs are involved in the accumulation of β-amyloid peptide in the brain in Alzheimer's disease.

  Non-Patent Document 7 discloses that accumulation of AGEs is related to kidney damage and retinopathy in diabetes. Aminoguanidine is known as a drug that inhibits the non-enzymatic saccharification reaction that is the basis for the accumulation of AGEs. Non-Patent Document 8 discloses that this aminoguanidine has been confirmed in model animals to be effective for diabetic complications such as retinopathy, kidney damage, and neuropathy. However, side effects such as headache and nausea have been reported for aminoguanidine in clinical trials as a treatment for diabetic complications, and there is a problem that long-term use is difficult.

  From such a background, development of a saccharification inhibitor derived from a natural product with few problems of side effects is expected. In addition, foods and drinks containing proteins and sugars have a problem that browning or precipitation occurs due to a non-enzymatic saccharification reaction during storage. As a method for solving this problem, for example, a method of using a sugar alcohol as a saccharide that is a raw material for foods and drinks can be considered. However, this method has a problem that the raw material to be used is limited.

As a drug for treating or preventing diseases related to AGEs by inhibiting the formation of AGEs in vivo, for example, Patent Document 1 discloses an extract of cultured cell extract of Murasaki spp. Maillard reaction inhibitors containing a polymer as an active ingredient are disclosed. Patent Document 2 discloses an AGE generation inhibitory composition containing a Maillard reaction inhibitor and vitamin B ₆ or a pharmaceutically acceptable salt thereof. Patent Document 3 discloses an agent for improving the carbonyl stress state in the abdominal cavity in peritoneal dialysis, which contains a carbonyl compound trapping agent (Maillard reaction inhibitor) as an active ingredient.

  Patent Document 4 discloses that rice bran and an aqueous extract of rice bran have specific binding activity to AGEs. Patent Document 5 discloses that an anti-glycation agent containing an almond extract suppresses the production of AGEs in the living body, and Patent Document 6 discloses that an anti-glycation agent containing a soybean extract suppresses the generation of AGEs in vivo. . Patent Document 7 discloses a cosmetic comprising, as an active ingredient, an extract of a camellia plant belonging to the lilium family (Liliaceae), and the effects of this cosmetic include elastase activity inhibitory action, antioxidant. It has also been disclosed to have an action, a protein saccharification inhibitory action, and a tyrosinase activity inhibitory action. Patent Document 8 discloses that an extract obtained by extracting cereal or a purified residue of cereal with a neutral medium reduces the tyrosinase activity of cultured pigment cells and suppresses pigmentation caused by melanin production or ultraviolet irradiation. As a specific example of this extract, a water extract from bran is mentioned in Preparation Example 6 of Patent Document 8.

  Patent Document 9 discloses that a hydrophilic solvent extract from barley has an antioxidant action and can effectively prevent oxidation of lipids contained in food. However, Patent Document 9 does not mention non-enzymatic saccharification reaction and AGEs, and does not disclose anything about the relationship between the solvent extract of wheat and non-enzymatic saccharification reaction or AGEs.

Bai P. et al., Conn Tissue Res1992; 28: 1-12 Sakata N. et al., Atherosclerosis 1995; 116: 63-75 Verzijl N. et al., Biochem J 2000; 350: 381-7 Prabhakaram M. et al., Mech Ageing Dev 1996; 91 (1): 65-78 Mizutari K. et al., J Invest Dermatol 1997; 108 (5): 797-802 Vitek MP. Et al., Proc Natl Acad Sci USA 1994; 91 (11): 4766-70 McCance DR. Et al., J Clin Invest 1993; 91: 2470-8 Brownlee M., Diabetes Care 1992; 15: 1835-43

JP 2008-214250 A Japanese Patent Laid-Open No. 10-324629 JP 2006-305345 A JP 2010-209003 A JP2012-72103A JP 2012-2119014 A JP 2011-225564 A JP 2004-83594 A Japanese Patent Laid-Open No. 10-140153

  An object of the present invention is to provide a non-enzymatic saccharification reaction inhibitor that suppresses a non-enzymatic saccharification reaction of a protein and is useful for solving various problems caused by the reaction.

  As a result of intensive research to solve the above-mentioned problems, the present inventors have found that an ethanol extract of wheat seeds such as wheat, barley, rye and oats suppresses non-enzymatic saccharification reaction of proteins, AGEs It has been found that the production of can be effectively inhibited.

  This invention is made | formed based on the said knowledge, and is a non-enzymatic saccharification reaction inhibitor which contains the solvent extract of wheat as an active ingredient.

  The non-enzymatic saccharification reaction inhibitor of the present invention suppresses a non-enzymatic saccharification reaction of a protein, and is useful for solving various problems caused by the reaction, for example, prevention / treatment of various symptoms or diseases. The non-enzymatic saccharification reaction inhibitor of the present invention is also useful for improving the quality of cosmetics, foods and drinks, and animal feeds. For example, during storage of cosmetics, foods and drinks and animal feeds containing proteins and sugars. It is possible to effectively prevent browning or precipitation due to non-enzymatic saccharification reaction of the protein, or to effectively suppress browning or precipitation that has occurred.

FIG. 1 is a graph comparing the degree of generation of AGEs (CML) with a control when various solvent extracts are added to a mixed system of protein and sugar.

  The non-enzymatic saccharification reaction inhibitor (hereinafter, also simply referred to as saccharification reaction inhibitor) of the present invention contains a solvent extract of wheat as an active ingredient. The wheat used as an extraction source in the present invention is usually an edible grass family. Specifically, for example, wheat such as bread wheat, durum wheat, crab wheat, spelled wheat, emma wheat, hard wheat, intermediate wheat, soft wheat (Gramineae), barley (Gramineae), rye ( (Gramineae) Examples include Amado Kumugi, Nogenashi Sukumugi and Boumgi, Inumugi belonging to the genus Vulgaris, and one of these can be used alone or in combination of two or more. Among these wheats, wheat, barley, rye and oat are particularly preferably used in the present invention because they are excellent in the non-enzymatic saccharification reaction inhibiting effect of the solvent extract.

  In the above wheat, the part used as the extraction source in the present invention is usually seeds such as wheat grains, barley grains, rye grains, oat grains and the like. The “seed” here includes embryo (embryo), endosperm, and bran (outer skin, seed coat). In the present invention, parts other than seeds (parts containing cell walls), for example, flowers, ears, floral patterns, stems, leaves, roots, and the like may be used as an extraction source. Seeds are most excellent in the enzymatic saccharification reaction inhibiting effect, and wheat seeds are preferably used as the extraction source.

  In the present invention, all of the wheat seeds (whole grain) may be used as the extraction source, and some of the wheat seeds (germ, endosperm, bran, etc.) may be used as the extraction source. When using a part of wheat seeds as an extraction source, bran (especially seed bran), especially wheat bran (bran), is particularly effective in suppressing non-enzymatic saccharification reaction of solvent extract. Are preferably used in the present invention. As bran (wheat bran), the remainder obtained by removing the endosperm from the seed (whole grain) produced by the normal milling process, or the germ from which the germ is further removed can be used. It doesn't matter.

  In the present invention, if necessary, the wheat can be crushed as a pretreatment for the extraction of the wheat (extraction source). In particular, when the extraction source is wheat seeds (whole grain), it is preferable to carry out a pulverization treatment before the extraction treatment with a solvent from the viewpoint of more reliably extracting the active ingredient. The method of the pulverization treatment is not particularly limited, and for example, the pulverization treatment can be performed using a known pulverizer such as a roll type, an airflow type, an impact type, or a barrel type. Furthermore, prior to the pulverization treatment, a pretreatment for increasing the efficiency of seed pulverization / extraction using an acid, an alkaline enzyme, or the like can also be performed. In addition, the wheat (extraction source) is usually a dried product, and when the pulverization is performed, the dried wheat is usually pulverized. The method for drying wheat is not particularly limited, and for example, a known drying method such as a heat drying method, an air drying method, a reduced pressure drying method, a freeze drying method, or a spray drying method can be used.

  Examples of the solvent used for the extraction process of wheat (extraction source) include water and organic solvents such as methanol, ethanol, acetone, etc., and one of these may be used alone or in combination of two or more. Can do. The organic solvent may contain water, and a water-containing organic solvent such as water-containing methanol, water-containing ethanol, water-containing acetone or the like can also be used as an extraction solvent. The content of the organic solvent in the water-containing organic solvent can be appropriately adjusted according to the type of extraction source. Among these extraction solvents, water, ethanol, and especially ethanol are particularly preferably used in the present invention because they are excellent in the non-enzymatic saccharification reaction inhibiting effect of the extract extracted from wheat using the solvent.

  When water is used as the extraction solvent, the hot water extraction method is preferred as the extraction method. Examples of the hot water extraction method include a method in which wheat (extraction source) is introduced into hot water. In a state where the extraction source is put into hot water, the extraction source may be fixed in hot water as it is (fixed by immersion), or the hot water may be stirred or refluxed. The temperature of the hot water is preferably 60 to 95 ° C, more preferably 80 to 95 ° C. On the other hand, when an organic solvent (including a water-containing organic solvent) such as methanol, ethanol, or acetone is used as the extraction solvent, the temperature of the solvent is usually set to be the same as room temperature (about 25 ° C.).

  The extraction time of wheat (extraction source) (the processing time of the extraction source with the extraction solvent) and the amount of the extraction solvent used can be appropriately selected according to the type of the extraction source and the extraction solvent, respectively. After extraction, the precipitate or insoluble fraction is removed by an appropriate separation means such as centrifugation or filtration, and the supernatant or soluble fraction is recovered. If necessary, the precipitate or insoluble fraction obtained by the first extraction process can be subjected to the same extraction process again to recover the supernatant or the soluble fraction. The target supernatant “solvent extract of wheat” is obtained by drying the collected supernatant or soluble fraction by a known drying method such as freeze drying, vacuum drying, or reduced pressure drying. .

  The wheat solvent extract thus obtained effectively inhibits the production of AGEs by suppressing the non-enzymatic saccharification reaction of the protein, as is apparent from the test results described in the following [Examples] section. Can do. Therefore, the saccharification reaction inhibitor of the present invention containing such a solvent extract as an active ingredient eliminates various inconveniences associated with the production of AGEs, for example, improves the quality of cosmetics, foods and drinks, and animal feeds ( For example, it is useful for prevention / suppression of browning or precipitation), or prevention / treatment of various symptoms or diseases. Examples of symptoms / disorders for which the glycation reaction inhibitor of the present invention is effective for prevention / treatment include diabetic complications, Alzheimer's disease, bone diseases, sagging / wrinkles of skin, and reduction of skin elasticity.

  Moreover, since the solvent extract of wheat is a component contained in wheat with abundant dietary experience, there is little concern that it will cause harmful effects on the living body even if ingested continuously for a long period of time. It is a substance. Therefore, the saccharification reaction inhibitor of the present invention containing such a solvent extract can be safely and continuously ingested not only by healthy persons and adults but also by elderly persons and sick persons. It is useful as a raw material for manufacturing them, as cosmetics, foods and drinks for humans, animal feeds, and the like. As used herein, “animal” includes mammals other than humans.

  In addition to the solvent extract, the pharmaceutical and cosmetic products may contain various pharmaceutically acceptable carriers, for example, excipients, stabilizers, other additives, and the like, if necessary. Alternatively, it may further contain other medicinal ingredients such as various vitamins, minerals, herbal medicines and the like. The pharmaceutical dosage form is not particularly limited, and includes all dosage forms that can contain the solvent extract. For example, tablets, capsules, granules, powders, syrups, dry syrups, liquids, Oral preparations such as suspensions; and parenteral preparations such as inhalants, transdermal preparations, suppositories, drops, and injections. Moreover, manufacture of the said pharmaceutical and cosmetics can be performed by the method normally used by those skilled in the art, and the compounding quantity of the said solvent extract, a compounding method, and a compounding time can be selected suitably. Content of the said solvent extract in the said pharmaceutical and cosmetics becomes like this. Preferably it is 0.05-5 mass%, More preferably, it is 0.5-5 mass%.

  Moreover, as said food / beverage products and animal feed, for example, a health food, a functional food / beverage product, a food / beverage product for specified health use, which is intended to have a protein saccharification reaction inhibitory effect (AGEs production inhibitory effect). Examples of foods and drinks for sick people, feeds for livestock, racehorses, appreciation animals, and pet foods. The form of the food and drink and animal feed is not particularly limited, and includes all forms that can contain the solvent extract, for example, it can be solid, semi-solid or liquid, or a tablet, chewable tablet, It can be in various forms such as powders, capsules, granules, drinks, gels, syrups, liquid foods for enteral feeding.

  Specific examples of the form of the food and drink include tea drinks such as green tea, oolong tea and tea, coffee drinks, soft drinks, jelly drinks, sports drinks, milk drinks, carbonated drinks, fruit juice drinks, lactic acid bacteria drinks, fermented milk drinks, powders Beverages, cocoa beverages, alcoholic beverages, purified water beverages, butter, jam, sprinkles, margarine spreads, mayonnaise, shortening, cream, dressing, breads, cooked rice, noodles, pasta, miso soup, tofu, milk , Yogurt, soups or sauces, confectionery (for example, biscuits and cookies, chocolate, candy, cake, ice cream, chewing gum, tablets) and the like. In addition, since animal feed can be utilized with a composition and form substantially the same as food and drink, the description regarding the food and drink in this specification is similarly applied to animal feed unless otherwise specified.

  In addition to the above-mentioned solvent extract, the above food and drink and animal feed are, if necessary, other food and drink materials, various nutrients and various kinds used for the production of general food and drink and animal feed that are usually eaten. Vitamins, minerals, amino acids, various fats and oils, various additives (for example, flavoring ingredients, sweeteners, acidulants such as organic acids, surfactants, pH adjusters, stabilizers, antioxidants, pigments, flavors), etc. You may contain. Moreover, manufacture of the said food-drinks and animal feed can be performed by the method normally used by those skilled in the art, and the compounding quantity of the said solvent extract, a compounding method, and a compounding time can be selected suitably. Content of the said solvent extract in the said food-drinks and animal feed becomes like this. Preferably it is 0.05-5 mass%, More preferably, it is 0.5-5 mass%.

EXAMPLES Hereinafter, although an Example and a test example are given and this invention is demonstrated further in detail, this invention is not restrict | limited by these Examples and a test example. Examples 1 to 5, 7 and 8 described later are reference examples.

[Examples and Comparative Examples]
Solvent extracts A-1 to A-7 and B-1 to B-5 were obtained from various extraction sources according to the following <Ethanol extraction treatment> or <Hot water extraction treatment>, and samples of each example and comparative example, respectively. It was. As an extraction source, the following plant seeds (dried seeds) were used. Wheat, barley, rye, oats (above, wheat), almonds, soybeans. For wheat, both the whole seed (whole grain) and its outer skin (wheat bran) were used as extraction sources, and for plants other than wheat, whole grain was used as the extraction source. When whole grain was used as the extraction source, the whole grain was pulverized prior to the extraction process. The pulverization process was performed by using a pulverizer (manufactured by Yasui Kikai, Multi Bead Shocker (registered trademark)) and performing a process of pulverizing whole grains for 10 seconds at 2500 rpm at room temperature (25 ° C.). did. When wheat bran was used as an extraction source, the pulverization process was not performed and the extraction process was performed as it was. The wheat bran was prepared according to a conventional method.

<Ethanol extraction treatment>
Add 15 ml of ethanol as extraction solvent to 3 g of extraction source, shake at room temperature (25 ° C.) for 2 hours, and then centrifuge at 5000 rpm under room temperature (25 ° C.) for 5 minutes to recover the supernatant. The supernatant was dried under reduced pressure to obtain an ethanol extract.
<Hot water extraction process>
15 ml of pure water as an extraction solvent is added to 3 g of the extraction source, the added pure water is heated to 80 ° C. to make hot water, and the extraction source is left in the hot water at 80 ° C. for 2 hours (fixed by immersion). Then, the mixture was centrifuged at 5000 rpm for 5 minutes to recover the supernatant, and the supernatant was dried under reduced pressure to obtain a hot water extract.

Example 1: Ethanol extract of wheat (A-1)
Example 2: Ethanol extract of wheat bran (A-2)
Example 3: Ethanol extract of barley (A-3)
-Example 4: Ethanol extract of rye (A-4)
-Example 5: Oat ethanol extract (A-5)
Example 6: Hot water extract of wheat bran (B-1)
-Example 7: Hot water extract of barley (B-2)
-Example 8: Hot water extract of oats (B-3)
Comparative Example 1: Almond ethanol extract (A-6)
Comparative Example 2: Soybean ethanol extract (A-7)
Comparative Example 3: Almond hot water extract (B-4)
Comparative Example 4: Soybean hot water extract (B-5)

[Test example: Confirmation test of saccharification reaction suppression effect]
Add 200 mM phosphate buffer (pH 7.4) to gelatin so as to be 4 mg / ml, swell for 30 to 60 minutes at room temperature, dissolve in a hot water bath at 50 to 60 ° C., and then filter with a 0.22 μm pore size filter. Filtered. Separately, 60 mM ribose was prepared with 200 mM phosphate buffer (pH 7.4), and then filtered through a filter having a pore size of 0.22 μm. Equal amounts of gelatin and ribose thus prepared are mixed to obtain a mixed system, and a test substance is added to the mixed system so that the final concentration is 5 mg / ml, and the test substance is not added (control). At the same time, it was allowed to stand at 37 ° C. for 1 week. As a test substance, luteolin was used as a positive control in addition to the above various solvent extracts. Luteolin was melted with ethanol and added to the mixed system so that the final concentration was 50 ppm (C-1) or 5 ppm (C-2). About the said mixed system (glycated gelatin) after leaving still for one week, CML was quantified by the following ELISA method using Nε- (carboxymethyl) lysine (CML) antibody which is a kind of AGEs, and the degree of CML formation was evaluated. The results are shown in FIG.

<ELISA method>
The saccharified gelatin which is the object of evaluation of the CML generation rate was diluted 1,800 times and 5,400 times with PBS. Further, 100 μl of the sample was dispensed into each well of the plate and allowed to stand at room temperature for 2 hours or overnight at 4 ° C. Then, after washing the sample three times with a washing solution, 200 μl of blocking solution is dispensed onto the sample, and allowed to stand at room temperature for 1 hour, and then the sample is washed three times with the washing solution, and then 100 μl of the primary antibody is dispensed onto the sample. The sample was washed with the washing solution three times, and then 100 μl of the secondary antibody diluted 10,000-fold with the washing solution was dispensed to the sample, and left at room temperature for 1 hour. Thereafter, the sample was washed three times with a washing solution, and then 100 μl of a color former was dispensed to the sample and allowed to develop color at room temperature. When the color gradation of the control appeared clearly, 100 μl of the reaction stop solution was dispensed into the sample to stop the reaction, the absorbance at 492 nm was measured, and the target CML was quantified.

Details of the reagents used in the above test are as follows.
・ Gelatin: Gelatin, from porcine skin type A (SIGMA G2500-100G) ・ Ribose: D (-)-Ribose (Wako Pure Chemical 180-01053).
Blocking solution: Gelatin hydrolysate Enzymatic diluted with PBS to give a 0.5% gelatin solution.
Primary antibody: CML: Anti Nε- (carboxylmethyl) lysine (736 μg / ml: distributed from Tokai University) diluted 1472 times with blocking solution.
Secondary antibody: Peroxidase-Labeled Antidoby to Mouse IgG (H + L) (KPL 074-1806). Add 500 μl of water to a vial (1 mg) and dissolve while spinning, then add 500 μl of glycerol and mix by pipetting to a concentration of 1 mg / ml.
Coloring agent: Use the one prepared by the following procedure. 100 ml of 100 mM citric acid and 200 ml of 100 mM disodium hydrogen phosphate are separately prepared, and the pH is adjusted to 5.0 while adding the latter to the former to obtain a citrate buffer. At room temperature, 10 ml of this citrate buffer and 1 tablet of OPD tablet (Wako Pure Chemicals 158-02151) are mixed, and 6 μl of 32% hydrogen peroxide solution (Kanto Chemical 18084-00) is added immediately before the color development test. Add the desired color former and use it.
Reaction stop solution: 36N H ₂ SO ₄ (Wako Pure Chemical Industries 199-15995) is diluted with ion-exchanged water to prepare 2N H ₂ SO ₄ for use.
-Cleaning solution: PBS (Dulbecco's composition: Nissui Pharmaceutical 08190) + 0.5% Tween 20 (Polyoxyethylene sorbitan monolaurate, Tokyo Chemical Industry 9005-64-5) is used.
・ Reaction plate: NUNC MAXISORP (NUNC 439454) is used.

  As is apparent from FIG. 1, the wheat solvent extracts A-1 to A-5 and B-1 to B-3 (Examples 1 to 8) were used as controls (negative control) to which no solvent extract was added. In comparison, the AGEs (CML) generation rate is low, and further, almond solvent extracts A-6 and B-4 (Comparative Examples 1 and 3) and soybean solvent extracts A-7 and B-5 (Comparative Example) Compared with 2 and 4), the AGEs (CML) generation rate was generally low. From this, it is clear that the solvent extract of wheat has a non-enzymatic saccharification reaction inhibitory effect of protein equivalent to or higher than the solvent extract of almond and soybean.

Claims (2)

A non-enzymatic saccharification reaction inhibitor containing a hot water extract of wheat bran as an active ingredient. The non-enzymatic saccharification reaction inhibitor according to claim 1 , wherein the hot water extract is an extract of hot water at 60 to 95 ° C.

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